Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis.
Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis.
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DOI：
10.1039/d1lc01053k
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发表时间：
2022-06-28
期刊：
影响因子：
6.1
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中科院分区：
文献类型：
--
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研究乳腺癌中的细胞和囊泡异质性仍然是一个挑战，这鼓励了模仿肿瘤微环境的可控体外系统的发展。虽然三维细胞培养更好地概括了在肿瘤生长和细胞外囊泡（EV）生物发生中观察到的异质性，但其生理相关性通常与二维细胞培养提供的对照形成对比。因此，为了挑战这种误解，我们开发了一种新型的微流体系统，该系统具有高度可调的三维EV微生物反应器（EVμ BR），以模拟乳腺癌中的微转移EV释放，同时利用微流体提供的方便，低体积和无菌界面。EVμ BR的直径和细胞占有率可以精确地定制为各种配置，支持乳腺癌肿瘤球体的形成。为了将EVμ BR包裹在微通道内并促进EV提取，利用基于数字微镜器件（DMD）的紫外（UV）投影系统，将自由基聚合中的氧抑制重新用于原位快速生成两层流体动力学陷阱。从微流控系统中收获少至20 μL的乳腺癌肿瘤球状体衍生的EV，并通过单EV免疫荧光对CD63和CD81进行定量。尽管低体积提取，但观察到单个EV上四跨膜蛋白的生物标志物表达和共表达的差异。此外，EVμ BR能够重现细胞和囊泡程度的异质性，表明微流体系统在乳腺癌和其他疾病模型中研究生理相关EV的实用性和稳健性。一种新的微流控系统，用于从封装在固定化水凝胶微生物反应器内的乳腺癌肿瘤球体中低体积收获细胞外囊泡，用于下游单囊泡分析。
Investigating cellular and vesicular heterogeneity in breast cancer remains a challenge, which encourages the development of controllable in vitro systems that mimic the tumor microenvironment. Although three-dimensional cell culture better recapitulates the heterogeneity observed in tumor growth and extracellular vesicle (EV) biogenesis, the physiological relevance is often contrasted with the control offered by two-dimensional cell culture. Therefore, to challenge this misconception we developed a novel microfluidic system harboring highly tunable three-dimensional EV microbioreactors (EVμBRs) to model micrometastatic EV release in breast cancer while capitalizing on the convenient, low-volume, and sterile interface provided by microfluidics. The diameter and cellular occupancy of the EVμBRs could be precisely tailored to various configurations, supporting the formation of breast cancer tumor spheroids. To immobilize the EVμBRs within a microchannel and facilitate EV extraction, oxygen inhibition in free-radical polymerization was repurposed to rapidly generate two-layer hydrodynamic traps in situ using a digital-micromirror device (DMD)-based ultraviolet (UV) projection system. Breast cancer tumor spheroid-derived EVs were harvested with as little as 20 μL from the microfluidic system and quantified by single-EV immunofluorescence for CD63 and CD81. Despite the low-volume extraction, differences in biomarker expression and coexpression of the tetraspanins on single EVs were observed. Furthermore, the EVμBRs were capable of recapitulating heterogeneity at a cellular and vesicular degree, indicating the utility and robustness of the microfluidic system to investigate physiologically relevant EVs in breast cancer and other disease models. A novel microfluidic system for the low-volume harvesting of extracellular vesicles from breast cancer tumor spheroids encapsulated within immobilized hydrogel microbioreactors for downstream single-vesicle analyses.
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